This invention relates to a biochemical separation or recovery process in which refractile bodies containing microbially produced proteins are separated or recovered from the microorganism hosts which produce them.
Many types of proteins, particularly proteins of potential therapeutic use such as interferon (IFN), interleukin-2 (IL-2), feline leukemia virus antigen (FeLV), etc. have been produced from transformed host cells containing recombinant DNA. The host cells are transformed with expression vectors containing genes encoding the desired heterologous protein and are then cultured under conditions favoring production of the desired protein.
Often the heterologous protein produced by the host cell precipitates inside the cell as opposed to being soluble in the cell. The intracellularly produced protein must be separated from the cellular debris and recovered from the cell before it can be formulated into a purified biologically active material.
Procedures for purifying native IL-2 from T cells are described by Watson, J. et al., J. Exp. Med. (1979) 150: 849-861; Gillis, S. et al., J. Immunology (1980) 124: 1954-1962; Mochizuki, D. Y., et al., J. Immunol. Meth. (1980) 39: 185-201; Welte, K. et al., J. Exp. Med. (1982) 156: 454-464; EP No. 92,163 and EP No. 94,317. In general, these procedures involve precipitating proteins from culture supernatants with ammonium sulfate followed by a chromatographic fractionation.
U.S. Pat. Nos. 4,450,103 and 4,462,940 and Derynck, R., et al., Nature (1980) 287: 193-197 describe procedues for recovering IFN-.beta. from IFN-.beta.-producing E. coli. The patents describe procedures in which IFN-.beta. is extracted from cellular material with 2-butanol or 2-methyl-2-butanol.
Commonly owned U.S. Pat. No. 4,569,790, filed Mar. 28, 1984 and issued Feb. 11, 1986 to K. Koths et al., describes a process for recovering IL-2 from an IL-2-producing microorganism whereby the microorganism cell membrane is disrupted, the disruptate is extracted with an aqueous solution of a chaotropic agent such as urea, the IL-2 is solubilized with, e.g., sodium dodecyl sulfate, and the IL-2 is separated in the presence of a reducing agent.
Commonly owned U.S. Pat. No. 4,530,787 filed Oct. 17, 1984 and issued July 23, 1985 to Z. Shaked et al., describes a process for oxidizing recombinant proteins such as IL-2 selectively and stoichiometrically using o-iodosobenzoic acid to ensure that the protein will be functionally equivalent to its native counterpart.
The above techniques for recovering the protein generally require use of costly reagents which must be removed from the protein prior to formulation thereof. Furthermore, many heterologous proteins are precipitated intracellularly in the form of refractile or inclusion bodies which appear as bright spots visible within the enclosure of the cell under a phase contrast microscope at magnifications down to 1000 fold. See. e.g., Miller et al., Science (1982) 215: 687-690 Cheng, Biochem. Biophys. Res. Comm., (1983) 111: 104-111. Using the techniques described above, such proteins may not be sufficiently isolated from protein contaminants or from forms of the protein which are biologically inactive when produced intracellularly.
Becker et al., Biotech. Advs. (1983) 1: 247-261 disclose separation of these bodies from most of the cell debris and soluble impurities by a low-speed centrifugation. In addition, Kleid et al., ch. 25 in Developments in Industrial Microbiology, Vol. 25, p. 317-325 (Society for Industrial Microbiology, Arlington, VA, 1984) disclose purification of refractile bodies by homogenization followed by centrifugation. Also, Marston et al., Bio/Technology (September, 1984), pp. 800-804 describe release of inclusion bodies by enzymatic and mechanical disruption procedures as well as sonication. Centrifugation at 12,000 xg for five minutes at 4.degree. C. of the cell lysates removed all the inclusion bodies from the supernatant. The resulting pellets are suspended in Triton X100 and EDTA and centrifuged before denaturation.
Purification and activity assurance of precipitated heterologous proteins is also described by U.S. Pat. Nos. 4,511,502; 4,511,503; 4,512,922; and 4,518,526; and EP No. 114,506. U.S. Pat. No. 4,511,502 describes purifying the refractile protein by isolation thereof from a host cell, dissolving the protein in a strong denaturing solution and removing impurities by high speed centrifugation. U.S. Pat. No. 4,511,503 describes and claims isolating the refractile protein from a host cell and treating the protein with a strongly denaturing solution. U.S. Pat. No. 4,512,922 describes and claims solubilizing the refractile bodies in a strongly denaturing solution and replacing with a weakly denaturing medium. U.S. Pat. No. 4,518,526 describes treating the host cell culture with a buffer of sufficient ionic strength to solubilize the host protein but not the refractile protein, disrupting the cells, and treating the insoluble fraction so as to obtain the refractile protein. EP No. 114,506 discloses a method for treating refractile material containing a heterologous protein so as to recover the protein from its host cell by contacting the refractile material with a denaturing solution which may optionally be contacted with a size-discriminating molecular sieve or subjected to high speed centrifugation to remove high molecular weight components from the solution. The examples of EP No. 114,506 indicate that the process requires repeated runs for successful recovery of product.
There remains a need in the art for a method of recovering refractile material containing heterologous expression products from the host cells which method is less costly, is easier to handle, and results in maximum recovery of pure protein in a biologically active form without use of chemical agents.